Insulin stimulates increased glucose transport into adipose and muscle tissue by causing translocation of the insulin responsive glucose transporter (GLUT4) from an intracellular pool to the plasma membrane. The inability of insulin's target tissues to respond in this manner is one of the primary defects in noninsulin-dependent diabetes mellitus. However, the signaling events that lead to GLUT4 translocation are poorly understood. The focus of this proposal is to investigate the intracellular insulin signaling pathways that trigger GLUT4 translocation. Elucidation of the mechanism whereby insulin stimulates glucose transport may identify new potential targets for therapeutic intervention in insulin resistant states. The experiments will be performed in 3T3-L1 adipocytes, in which insulin-stimulated GLUT4 translocation can be assessed on a single cell basis by immunofluorescence staining with anti GLUT4 antibodies. Microinjection of single living cells will be used to deliver various reagents to activate or inactivate specific intracellular molecules that may be involved in insulin action. Nearly any type of reagent can be delivered in this way, such as antibodies, peptides and recombinant proteins. There are many potentially involved signaling molecules that will be studied with this system, including substrates of the insulin receptor, p21 ras, the Raf, MEK and ERK Ser/Thr kinases, phosphatidylinositol 3-kinase and Rab small GTP-binding proteins.